Process for production of inhibited forms of activated blood factors

ABSTRACT

A process for producing a highly purified preparation of an inhibited form of an activated blood factor entails providing a partially purified preparation containing the blood factor of interest, treating the partially purified preparation to convert the blood factor to an inhibited activated form in a single step, and then purifying the resulting inhibited activated blood factor.

This application is a continuation of application Ser. No. 08/330,978,filed Oct. 28, 1994.

FIELD OF THE INVENTION

This invention relates to the production of blood factors, andparticularly this invention relates to large-scale production ofpurified inhibited forms of activated blood factors.

BACKGROUND OF THE INVENTION

Following initiation of the clotting process, blood coagulation proceedsthrough the sequential activation of certain plasma proenzymes to theirenzyme forms. These plasma glycoproteins, including Factor XII, FactorXI, Factor IX, Factor X, Factor VII, and prothrombin, are zymogens ofserine proteases. Most of these blood clotting enzymes are effective ona physiological scale only when assembled in complexes on membranesurfaces with protein cofactors such as Factor VIII and Factor V. Otherblood factors modulate and localize clot formation, or dissolve bloodclots. Activated protein C is a specific enzyme that inactivatesprocoagulant components. Calcium ions are involved in many of thecomponent reactions. Blood coagulation follows either the intrinsicpathway, where all of the protein components are present in blood, orthe extrinsic pathway, where the cell-membrane protein tissue factorplays a critical role. Clot formation occurs when fibrinogen is cleavedby thrombin to form fibrin. Blood clots are composed of activatedplatelets and fibrin.

Thrombin is a multifunctional protease that regulates several keybiological processes. For example thrombin is among the most potent ofthe known platelet activators. In addition, as noted above, thrombin isessential for the cleavage of fibrinogen to fibrin to initiate clotformation. These two elements are involved in normal hemostasis but inatherosclerotic arteries can initiate the formation of a thrombus, whichis a major factor in pathogenesis of vasoocclusive conditions such asmyocardial infarction, unstable angina, nonhemorrhagic stroke andreocclusion of coronary arteries after angioplasty or thrombolytictherapy. Thrombin is also a potent inducer of smooth cell proliferationand may therefore be involved in a variety of proliferative responsessuch as restenosis after angioplasty and graft induced atherosclerosis.In addition, thrombin is chemotactic for leukocytes and may thereforeplay a role in inflammation, Hoover, R. J. et al., Cell 14:423 (1978);Etlngin, O. R. et al., Cell 61:657 (1990). These observations indicatethat inhibition of thrombin formation or inhibition of thrombin itselfmay be effective in preventing or treating thrombosis, limitingrestenosis and controlling inflammation.

The formation of thrombin is the result of the proteolytic cleavage ofits precursor prothrombin at the Arg-Thr linkage at positions 271-272and the Arg-Ile linkage at positions 320-321. This activation iscatalyzed by the prothrombinase complex, which is assembled on themembrane surfaces of platelets, monocytes, and endothelial cells. Thecomplex consists of Factor Xa (a serine protease), Factor Va (acofactor), calcium ions and the acidic phospholipid surface. Factor Xais the activated form of its precursor, Factor X, which is secreted bythe liver as a 58 kDa precursor and is converted to the active form,Factor Xa, in both the extrinsic and intrinsic blood coagulationpathways. It is known that the circulating levels of Factor X, and ofthe precursor of Factor Va, Factor V, are on the order of 10-7M. Themhas been no determination of the levels of the corresponding activeFactors Va and Xa.

The amino acid sequences and genes of most of the plasma proteinsinvolved in hemostasis of blood are commonly known, such as Factor IIa,Factor Va, Factor VIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa,Activated Protein C, Activated Protein S, fibrinogen and thrombin. Alsocommonly known am the amino acid sequences and genes of the precursorforms of these blood factors, and common methods for their activation orconversion to mature forms.

Factor X (Stuart Factor) is an essential component of the bloodcoagulation cascade (see, FIGS. 1 and 2). Factor X is a member of thecalcium ion binding, gamma carboxyglutamyl ("Gla")-containing, vitamin Kdependent, blood coagulation glycoprotein family, which also includesFactors VII and IX, prothrombin, protein C and protein S, Furie, B. etal., Cell 53:505 (1988). Factor X is the zymogen for the serine proteaseFactor Xa. Factor Xa combines with a co-factor, activated Factor V,calcium, and phospholipids on a membrane surface to form theprothrombinase complex. This enzyme complex converts prothrombin tothrombin, which then converts fibrinogen to fibrin, one of the pathwaysresulting in thrombosis (Colman, R. W. et al., "Overview of Hemostasis"in Colman, R. W., et al., Hemostatis and Thrombosis, Basic Principlesand Clinical Practice, Second Edition (1987), Part I, Section A, PlasmaCoagulation Factors, pp. 3-17).

Factor X can be purified from natural, synthetic or recombinant sourcesby any of a number of different extractive and chromatographictechniques, such as: a combination of ion-exchange, heparin-affinity andhydroxylapatite chromatography (Kosow, D. P., Thromb. Res., 9(6):565-573(1976); sulfated dextran (Miletich, J. P. et al., AnalyticalBiochemistry 105:304-310 (1980)); a combination of barium citrateadsorption, ammonium sulfate precipitation, ion-exchange andheparin-affinity chromatography (Bajaj, S. P. et al., Prep. Biochem11:397-412 (1981)); Cohn fractionation (Monohan, J. B. et al., Thromb.Res. 19(6):743-755 (1980)); sulfated non-carbohydrate matrices (U.S.Pat. Nos. 4,721,572; and 4,725,673); immunoaffinity chromatography(European Patent Application 0 286,323); hydrophobic interactionchromatography (Freidberg, R. C., et al., Prep. Biochem. 18(3):303-320(1988) ); metal-chelate chromatography (PCT/GB88/01150); a combinationof immunoaffinity and ion-exchange (Ahmad, S. S. et al., Thromb. Res.55(1):121-133 (1989)); and as a by-product in the purification of otherblood coagulation factors (Hrinda, M. E., et al., "Preclinical Studiesof a Monoclonal Antibody-Purified Factor IX; Mononine™," in Seminars inHematology 28(3) Suppl. 6:6-14 (1991); and U.S. Pat. No. 5,071,961.)Typically, Factor X activation, inactivation and purification areaccomplished separately.

Factor X must be activated to Factor Xa before the protease isincorporated into the prothrombinase complex (Steinberg, M. et al.,"Activation of Factor X" in Colman, R. W. et al., supra, Part I, SectionA, Chapter 7, pp 112-119). Factor Xa is a two chain molecule linked byone disulfide bond between the two chains. The heavy chain contains theserine protease, trypsin-like active site and the N-terminal activationpeptide which is glycosylated. The heavy chain has at least three forms,a, β and g, which differ due to the cleavage of a C-terminal peptide inthe heavy chain (Aronson, D. L. et al., Proc. Soc. Exp. Biol. Med.137(4):1262-1266 (1971); Mertens, K. et al., Biochem J. 185:647-658(1980)). This C-terminal peptide is thought to be glycosylated throughan O-linked type glycosylation. The a form is the full length form ofthe heavy chain and the β and g forms are clipped. The light chaincontains a growth factor-like domain and a number of uniquepost-translationally modified amino acid residues, called gamma-carboxyglutamic acid residues ("GLA's") which are implicated in impartingactivity through calcium binding interactions required in theprothrombinase complex (Davie, E. W., "The Blood Coagulation Factors:Their cDNAS, Genes and Expression" in Colman, R. W. et al., supra, PartI, Section A, pp. 242-268).

Factor X can be activated to Factor Xa by any of several methods. FactorX is activated naturally through the extrinsic pathway (FactorVIIa/Tissue Factor complex) or the intrinsic pathway (FactorVIIIa/Factor Ixa-phospholipid-calcium enzyme complex) (Mertens, K. etal., Biochem J. 185:647-658 (1980); Jesty, J., J. Biol. Chem.261(19):8695-8702 (1986); Steinberg, M. et al., supra; Bauer, K. et al.,Blood 74(6):2007-2015 (1989); Chattopadhyay, A. et al., J. Biol. Chem.2:735-739 (1989)). Factor X can also be activated to Factor Xa byproteases such as Russell's Viper Venom Factor X activating enzyme("RVV-X") (Furie, B. C. et al., Methods in Enzymology 45:191-205 (1976);DiScipio, R. G. et al., Biochemistry 16(24):5253-5260 (1977); trypsin(Steinberg, M., et al., supra); or cancer procoagulant (Gordon, S. G. etal., Blood Coagulation and Fibrinolysis 2:735-739 (1991)).

It is known that numerous snake venom activities affect the intrinsiccoagulation mechanism by variously activating, inhibiting or convertingfactors in the blood coagulation cascade; snake venoms are known whichactivate Protein C, prothrombin, thrombin-like enzymes, fibrinogenases,and activities of Factors V and X (N. A. Marsh, Blood Coagulation andFibrinolysis 5:399-410 (1994). Synthetic peptides and peptidomimeticsare also known as substrates and inhibitors of serine proteases(Claeson, G., Blood Coagulation and Fibrinolysis 5:411-436 (1994). Anumber of general and specific serine protease inhibitors are alsoknown.

Various activators and inhibitors are commonly known for many of theblood factors. For example, Factor I (fibrinogen) is known to beactivated by thrombin; Factor II (prothrombin) is known to be activatedby Factor Xa and thrombin; Factor V is known to be activated by papain,a Factor-V-activation protease from Russell viper venom, plasmin, FactorXa, chymotrypsin, and thrombocytin, and is inactivated by activatedProtein C; Factor VII is known to be activated by minor proteolysis,with a signal peptidase and a processing protease; Factor IX is known tobe activated by Factor XIa with calcium ions, tissue factor, Factor VII,and Russell viper venom-X, and is known to be inactivated by hirudin andantithrombin III; Factor X is activated by Factors IXa and VII withphospholipid and calcium ions, and by Russell viper venom; Factor XI isknown to be activated by Factor XIIa and trypsin; Factor XII is known tobe activated by contact with negatively charged surfaces, sulfatides,trypsin, plasmin, and kallikrein; Protein C is activated by thrombin,etc. See, Colman et al., supra. for the text describing known bloodfactor activators and inactivators.

In some circumstances, it is desirable to interfere with the functioningof Factor Xa in order to prevent excessive clotting. In othercircumstances, such as in hemophilia, it is desirable to provide asource of Factor Xa independent of the activation process that takesplace in normal individuals. Both of the common forms of hemophilia(hemophilia A and B) involve deficiencies in only the intrinsic pathwayof activation, but the operation of the extrinsic pathway does notappear to be successful in arresting bleeding. Similarly, other patientsare treated currently for deficiencies of other blood factors (such asVII, X, XI, XIII), or yon Willebrand's disease. Factor VII deficiency isnot as clinically well-defined as hemophilia A or B, however patientswith Factor VII deficiency have been reported to have extensivebleeding. Protein C deficiency is associated with thrombotic risk.

Factor Xa, and several other activated blood factors, have typically notbeen useful as pharmaceuticals because of their extremely shorthalf-life in serum, which for example typically is only about 30 secondsfor Factor Xa. Use of acylation to prolong the half-life of certainblood factors has been disclosed. For example, Casseis, R. et al.,Biochem. Jour. 247:359-400 (1987), reports that various acylating agentsremained bound to urokinase, tPA and streptokinase-plasminogen activatorcomplex for time periods ranging from a half-life of 40 minutes to ahalf-life of over 1,000 minutes depending on the nature of the acylatinggroup and the nature of the factor. U.S. Pat. No. 4,337,244 describesacylation of tPA or streptokinase. Use of an amidinophenyl groupfunctioning as an arginine analog to introduce, temporarily, asubstituted benzoyl group into the active site for the purpose ofenhancing serum stability was discussed by Fears, R. et al., Seminars inThrombosis and Homeostasis 15:129-39 (1980) (see also: Fears, R. et al.,Drugs 33 Suppl. 3:57-63 (1987); Sturzebecher, J. et al., Thrombosis Res.47:699-703 (1987)), which describes stabilized acyl derivatives of tPA.Use of the acylated plasminogen streptokinase activator complex("APSAC") is described in Crabbe, S. J. et al., Pharmacotherapy10:115-26 (1990). Acylated forms of thrombin have also been described.Generally, methods for activating, inhibiting, and recovering the targetblood factor have been multi-step and complex processes.

Chemically inactivated forms of Factor Xa can be used in a number oftherapeutic indications (U.S. Pat. Nos. 4,285,932; 5,120,537; Benedict,C. R. et al., Blood 81(8):2059-2066 (1993); U.S. Ser. No. 08/268,003,filed Jun. 26, 1994; Sinha, U. et al., "Procoagulation Activities ofReversibly Acylated forms of Factor Xa," presented at the 35th AnnualMeeting of the American Heart Association, St. Louis, Mo., Dec. 3-7,1993). Factor Xa can be irreversibly inactivated using chloromethylketone derivatives, such as glutamyl glycyl arginyl ("EGR") chloromethylketone, or dansyl glutamyl glycyl arginyl ("DEGR") chloromethyl ketone(see e.g.: Nesheim, H. E. et al., Jour. Biol. Chem. 254:10952 (1979);U.S. Pat. No. 5,120,537; Kettner, C. et al., Biochem 17(22):4778-4783(1978); Kettner, C. et al., Biochim. Biophys. Acta. 569:31-40 (1979);Kettner, C. et al., Arch. Biochem. Biophys. 202:420-430 (1980); Kettner,C. et al., Methods in Enzymology 80 Part C:826-842 (1981); Kettner, C.et al., Thromb. Res. 22:645-652 (1981); Nesheim, M. E. et al., J. Biol.Chem. 256(13):6537-6540 (1981); U.S. Pat. No. 4,318,904; Lijnen, H. R.et al., Thromb. Res. 34:431-437 (1984); Williams, B., et al., J. Biol.Chem. 264(13):7536-7545 (1989); U.S. Pat. No. 5,153,175). Thisirreversibly inactivated Factor Xa can be used to inhibit thrombingeneration in-vivo and thus be utilized as an anticoagulant (U.S. Pat.No. 5,120,537, and Benedict, C. R. et al. supra).

Factor Xa can be reversibly inactivated using various derivatives of4-amidinophenyl benzoate (or p-amidinophenyl ester HCl) acylatingcompounds which impart reversibility at varying rates. This reversiblyinactivated Factor Xa can be used to promote thrombin formation in vivoand thus can be utilized in procoagulant indications (U.S. Pat. No.4,285,932; U.S. Ser. No. 08/268,003, filed Jun. 26, 1994; and Sinha etal., supra).

SUMMARY OF THE INVENTION

The invention features a process for producing a highly purifiedpreparation of an inhibited (that is, inactivated permanently ortransiently) form of an activated blood factor, by providing a partiallypurified preparation containing the blood factor, treating the partiallypurified preparation to convert the blood factor to an inhibitedactivated form in a single step (and/or in a single reaction vessel),and then purifying the resulting inhibited activated blood factor.

The invention provides for production of activated blood factors inpermanently or transiently inhibited form, at high purity and in highyield. In certain embodiments, the methods of this invention can be usedto prepare inhibited activated Factor II (inhibited Factor IIa),inhibited activated Factor V (inhibited Factor Va), inhibited activatedFactor VII (inhibited Factor VIIa), inhibited activated Protein C,inhibited activated Protein S, inhibited activated Factor IX (inhibitedFactor IXa), inhibited activated Factor X (inhibited Factor Xa),inhibited activated Factor XI (inhibited Factor XIa), inhibitedactivated Factor XII (inhibited Factor XIIa), and inhibited activatedfibrinogen (inhibited Factor I).

The inhibition treatment can immediately follow the activatingtreatment, with or without an intervening process step, or, theactivation and inhibition treatments can be carried out concurrently.

The partially purified preparation, containing the blood factor, can bederived from natural, synthetic or from recombinant source materials.

In some embodiments the inhibition treatment includes using a peptidylchloromethyl ketone derivative, preferably being tri-peptidyl orgreater, such as EGR-ck or DEGR-ck.

In some embodiments the inhibition treatment includes causing an acylgroup to be bound at the active site of a blood factor (in activated orzymogen form), where it inhibits clearance and is susceptible to slowhydrolysis to generate the active form of the blood factor, resulting ina reversibly inhibited activated blood factor.

Other features and advantages will be apparent from the specificationand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (SEQ ID NO:1 and SEQ ID NO:2) is a schematic showing a humanFactor X, indicating regions of the molecule.

FIGS. 2A (SEQ ID NO:3) and 2B (SEQ ID NO:4) are schematics showing,respectively, a and 13 forms of a human Factor Xa.

FIG. 3 is a schematic block diagram showing process steps according toan exemplary embodiment of the invention for producing an inhibitedactivated blood factor.

DESCRIPTION OF SPECIFIC EMBODIMENTS Definitions of Terms

As used herein, the terms "blood coagulation factor" and "blood factor"mean and refer to blood factors generally, and particularly to any of anumber of peptides, factors and cofactors, which comprise the intrinsicor extrinsic blood coagulation cascade in humans, or are involved inmodulation, localization or dissolution of blood clots. Blood factorssuitable for use in this invention include, but are not limited to,Factors II, V, VII, IX, X, XI and XII, Proteins C and S, thrombin,fibrinogen, etc., in their zymogen, non-activated, activated orinhibited activated forms. The term "blood factor" refers to therespective native, synthetic or recombinantly produced polypeptidesequence as commonly known.

As used herein, the term "impure starting protein fraction" refers toany protein fraction either from natural, synthetic or recombinantsources which contains the blood factor of interest in combination withother proteins, or in combination with other materials present in theenvironment wherein the protein fraction was produced or derived.

The term "partially purified preparation" means a preparation thatcontains a blood factor of interest and that is substantially orcompletely free of inhibitors of the blood factor of interest. Incertain embodiments, the partially purified preparation is substantiallyfree of chelating agents or contains free calcium in molar excess of anychelating agents that may be present. A partially purified preparationmay at be at a high level of purity.

"Factor X" refers to the native, synthetic or recombinantly producedsingle- or two-chain Factor X sequence, essentially as shown in FIG. 1or FIG. 2, containing at a minimum the heavy chain to which is attachedthe activation peptide, at its N-terminus, and the light chain. Thesemay or may not be linked through a cleavage sequence as indicated in thefigures.

"Factor IX", "Factor VII" and "Protein C" refer to the respective nativeor recombinantly produced protein sequence as commonly known.

The terms "chemical inhibitor" and "chemical inactivator", as usedherein, mean and refer to any of a number of reactive peptidyl ororganic molecules which have the ability to covalently bind to theactive site of the activated blood coagulation factor and to render theactivated blood coagulation factor inactive, that is, to inhibit theactivity of the activated blood factor. Known reactive compounds includetri- (or greater-) peptidyl chloromethyl ketone derivatives or tri- (orgreater-) peptidyl arginyl chloromethyl ketones to produce irreversiblyinhibited compounds or any of a group of acylating agents which canproduce transiently inhibited blood factors.

A blood factor that is "activated", as that term is used herein, is onethat has been catalytically formed from an inactive zymogen precursor.

An activated blood factor that is "inhibited", as that term is usedherein, is one that substantially lacks the enzymatic activity expectedfor the blood factor when activated.

"Factor Xa" refers to native, synthetic or recombinantly produced,enzymatically active Factor X containing light and heavy chain only. Theactivation peptide is not present in this complex.

"Inhibited Factor Xa" means and refers to a modified form of Factor Xawhich is activated in the sense that it combines to form theprothrombinase complex, but which has no serine protease activity byvirtue of the modification of its active site.

"Acylated Factor Xa" or "AcXa", unless otherwise specified, refers toFactor Xa, whether produced recombinantly or not, wherein the serinecatalytic domain has been blocked with a substituent which provides theAcyl-Factor Xa with a half-life in serum of at least 5-10 minutes,preferably more than 15 minutes, and which releases Factor Xa in activeform over this time period. The half-life in serum can be measureddirectly in vivo using a suitably labeled form. However, it ispreferable to assess the ability of the extended life AcXa to generatethe active factor Xa within the required time frame in vitro using as acriterion in vitro assays for which Xa is a catalyst. Under theseconditions, suitable forms of Acylated Factor Xa for the inventioninclude those which have a rate constant for hydrolysis in isotonicaqueous media at pH 7.4 and 37° C. such that a half-life ofapproximately 5 minutes to several hours is achieved. The half-life canbe determined directly in vitro by measuring the rate of hydrolysis ofthe acylated Xa, if desired, using its ability to activate clotting, orthe prothrombinase reaction as criteria for Xa formation.

The blood factors described in this invention are defined herein to beany isolated polypeptide sequence which possesses a biological propertyof the naturally occurring blood factor polypeptide comprising acommonly known polypeptide sequence, variants and homologues thereof,and mammalian or other animal analogues.

"Biological property" for the purposes herein means an in vivo effectoror antigenic function or activity that is directly or indirectlyperformed by a blood factor (whether in its native or denaturedconformation), or by any subsequence thereof. Effector functions includereceptor binding, any enzyme activity or enzyme modulatory activity, anycarrier binding activity, any hormonal activity, any activity inpromoting or inhibiting adhesion of cells to an extracellular matrix orcell surface molecules, or any structural role. However, effectorfunctions do not include antigenic functions, i.e., possession of anepitope or antigenic site that is capable of cross-reacting withantibodies raised against a naturally occurring blood factorpolypeptide.

Ordinarily, the blood factors claimed herein will have an amino acidsequence having at least 75% amino acid sequence identity with acommonly known sequence, most preferably at least 80%, even morepreferably at least 90%, and most preferably at least 95%. Identity orhomology with respect to a commonly known blood factor sequence isdefined herein as the percentage of amino acid residues in the candidatesequence that are identical with the known blood factor amino acidresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology, and not consideringany conservative substitutions as part of the sequence identity. None ofN-terminal, C-terminal or internal extensions, deletions or insertionsinto the blood factor sequence shall be construed as affecting homology.

Thus, permanently or transiently inactivated blood factor polypeptidesand blood factors with extended plasma half-lives that can be madeaccording to this invention include each blood factor sequence;fragments thereof having a consecutive sequence of at least 5, 10, 15,20, 25, 30 or 40 amino acid residues from a commonly known blood factorsequence; amino acid sequence variants of a commonly known blood factorsequence wherein an amino acid residue has been inserted N- orC-terminal to, or within, the blood factor sequence or its fragment asdefined above; amino acid sequence variants of the commonly known bloodfactor sequence or its fragment as defined above has been substituted byanother residue. Blood factor polypeptides include those containingpredetermined mutations by, e.g., site-directed or PCR mutagenesis, andother animal species of blood factor polypeptides such as rabbit, rat,porcine, non-human primate, equine, murine and ovine blood factors, andalleles or other naturally occurring variants of the foregoing and humansequences; derivatives of the commonly known blood factor or itsfragments as defined above wherein the blood factor or its fragmentshave been covalently modified by substitution, chemical, enzymatic orother appropriate means with a moiety other than a naturally occurringamino acid (for example, a detectable moiety such as an enzyme orradioisotope); glycosylation variants of the blood factor (insertion ofa glycosylation site or deletion of any glycosylation site by deletion,insertion or substitution of appropriate amino acid); and soluble formsof the blood factor.

MODES OF CARRYING OUT THE INVENTION General

As summarized above, this invention provides a process for producinglarge-scale quantities of chemically inactivated (i.e., chemicallyinhibited) activated blood factors from an impure starting proteinfraction. Generally, the process includes one or more steps to obtain apartially purified preparation containing the blood factor of interest;the step of treating the partially purified preparation to activate andinhibit the blood factor; and steps to complete the purification of theresulting inhibited activated blood factor. The inhibition treatment canimmediately follow the activating treatment, with no intervening processstep; or, the activation and inhibition treatments can be carried outconcurrently.

FIG. 3 shows a block diagram outlining a preferred embodiment of theprocess of this invention for producing irreversibly or reversiblyinhibited forms of blood coagulation factors, with specific reference toFactor X inactivated with EGR-ck.

Referring to the preferred embodiment exemplified in FIG. 3, thestarting material is a plasma fraction, preferably virally inactivated,containing the blood factor of interest. The starting material mayalternatively be a product of recombinant expression of the bloodfactor. The starting material may be initially processed, for examplethrough an affinity purification chromatography column (e.g., animmunoaffinity column), to produce the partially purified preparationcontaining the blood factor of interest. As shown in FIG. 3, a highlyspecific affinity purification step is used, so that the resultingelution pool contains the desired blood coagulation factor at a highlevel of purity.

The partially purified preparation may then be concentrated and/ordiafiltered into a buffer suitable for carrying out the activation andinactivation (inhibition) treatments. In FIG. 3, the blood factor ofinterest is Factor X, which can be activated using RVV-X, purified fromthe venom of Vipera russelli, to produce Factor Xa; and Factor Xa can beinactivated using a peptidyl chloromethyl ketone or an acylating agent.Here, the preparation is treated concurrently with RVV-X and EGR-ck, toproduce EGR-Factor Xa.

Thereafter a series of final purification steps is carried out to bringthe inhibited activated blood factor of interest to a desired level ofpurity. Particularly, as in the example in FIG. 3, the treatedpreparation may subjected to a further viral clearance step, an ionexchange step to remove various contaminants and, optionally, anadditional inactivation step (here using EGR-ck) to sweep upsubstantially all remaining activated factor. The product may then beconcentrated and diafiltered into a storage buffer.

Partial Purification of Source Material Containing the Blood Factor

Any of a variety of techniques and combinations of techniques, known inthe art, may be used to partially purify the preparation to make itready for activation and inhibition treatment. Preferably the partiallypurified preparation contains substantially no inhibitors of the bloodfactor of interest; and preferably it contains no blood coagulationfactors other than the factor of interest, although other zymogenfactors may be present. For example, where Factor X is the blood factorof interest, and Russell viper venom (RVV-X) is used as an activatingagent, the partially purified preparation should be substantially freeof Factors V and IX, as Factors V and IX are also subject to activationby RVV-X. Where the blood factor is calcium-dependent, the use ofchelators should avoided, unless free calcium is present in a molarexcess of the chelator. For this reason, EDTA and EGTA buffers are lesspreferred.

Preferably (for improved yield), although not necessarily, the bloodfactor of interest is present in the partially purified preparation atabout 50% purity, more preferably at about 80% purity, and still morepreferably at about 90% purity. Preferred techniques for partialpurification include, for example, column chromatographic techniquesusing immunoaffinity, heparin-affinity, and hydroxylapatite, sulfateddextrans, ion-exchange chromatography, metal-chelate chromatography,sulfated non-carbohydrate matrices, Cohn fractionation, hydrophobicinteraction chromatography ("HIC"), and ammonium sulfate precipitation.DEAE resins are suitable, and preferably (although not necessarily)anion exchange chromatography can be used, also preferred are any ofvarious quaternary amine columns can be used, e.g., the "Q" columns.

In certain preferred embodiments, an immunoaffinity resin is preparedand used according to generally accepted methods in the field. Preferredresins include Tresyl-activated Agarose, under the registered trademarkAffinica® from Schleicher and Scheull, as well as other Tresyl activatedresins, Aldehyde activated resins, Triazine activated resins, Hydrazideactivated resins, Azlactone activated resins, and others. Typically,using standard techniques, a hybridoma cell line producing a monoclonalantibody with specificity for the target blood factor is obtained. Thecell line is then injected into mice in order to conveniently producequantities of the monoclonal antibody in ascites fluid, howeverrecombinant production or other antibody/antibody fragment productiontechniques may advantageously be utilized.

The monoclonal antibody may then be chromatographically purified usingstandard techniques such as protein A and ion-exchange chromatographytechniques to greater than 98% purity. The required amount of resin maybe prepared according to the manufacturer's instructions and both theresin and antibody may then be buffer exchanged into the couplingbuffer. In a preferred embodiment, the coupling buffer contained 0.1Msodium carbonate at pH 8.5, and the antibody solution was incubatedovernight with the Tresyl-activated resin at 2°-8° C. to allow efficientantibody coupling. The antibody can be coupled to the resin according tomethods known in the art, commonly at ratios of between 1-10 mg antibodyper milliliter of resin. After the coupling step, the linked resin iswashed and blocked, typically according to manufacturers instructions,and packed into an appropriate chromatography column (either radial oraxial flow geometries) for use in the purification of the blood factorof interest.

In a particularly preferred embodiment, an anti-Factor X immunoaffinitycolumn is set up using an immunoaffinity resin made as described aboveand used to partially purify a plasma fraction containing Factor X asthe blood factor of interest. A frozen Factor X containing plasmafraction (e.g., a Factor IX affinity chromatography column washfraction, or a DEAE or calcium phosphate eluate from a plasmafractionation process, Cohn fraction, etc.) is obtained; the plasmafraction may have been heat or solvent-detergent treated to reducepotential viral load and pH adjusted to neutral pH ±0.5 units. Theplasma fraction is thawed to between 2° and 8° C., 0.2 μm filtered andapplied to an anti-Factor-X immunoaffinity column equilibrated withphosphate buffered saline solution at 2°-8° C. The residence time of theload through the column is set to be greater than or equal to 5 minutes.Once an appropriate load for the binding capacity of the column(generally 0.1-1.0 mg antigen/ml resin) has been applied, the column iswashed with a volume of phosphate buffered saline equal to at least 10column volumes. After sufficient column washing, the purified antigen,Factor X, is eluted using 0.1M CAPS buffer containing 25 mM sodiumchloride, pH 10.5-11.3. The elution pool which is >90% Factor X isimmediately titrated to neutral pH ±0.5 units with concentrated (2-3M)HEPES buffer.

The Activation and Inhibition Step

In the activation and inhibition step, an inhibition (inactivation)treatment is carried out concurrently with an activation treatment; oran inactivation treatment follows an activation treatment with orwithout intervening processing steps. Techniques for activating andinactivating any of the various blood factors are known in the art andare discussed above by way of background.

Irreversible Inactivation

Generally, irreversible inactivation may be accomplished by any of avariety of methods discussed above, including irreversible inactivationby chloromethyl ketone derivatives, or by using small molecules whichcovalently and irreversibly bind to the active site of the blood factor.

In particularly preferred embodiments, a preparation containing FactorX, purified as described above, can be treated to activate and inhibitthe Factor X as follows. The purified Factor X is concentrated toapproximately 1 mg/ml utilizing a Filtron ultrafiltration system with 8kDa MWCO Omega type membranes, or another equivalent system. Theconcentrated Factor X can then be diafiltered into 50 mM Tris buffer, 25mM sodium chloride, pH 7.5 or can be directly activated, without bufferexchange, using Russell's Viper Venom Factor X activating enzyme (RVV-X)at a mass:mass ratio of Factor X:RVV-X of between 1000:1 to 20:1 at18°-37° C. in the presence of 5 mM calcium chloride for at least 5-10minutes. Purified RVV-X can be prepared through a number of previouslypublished processes (Williams, W. J. et. al, Biochem. J. 84:52-62(1962); Kisiel, W. et al., Biochem. 15(22):4901-4906 (1976); and Takeya,H. et al., J. Biol. Chem. 267(20):14109-14117 (1992)) from crude RVV.The reaction may be stopped after one hour with the addition of EDTA to10 mM. The activated Factor X (Factor Xa) can be either simultaneouslyor sequentially reacted with a covalent inhibitor, either a tripeptidechloromethyl ketone or acylating agents (e.g., variants of4-amidinophenyl benzoate or others as discussed below), at a molar ratioof greater than 20:1 inhibitor:Factor X for at least 30 minutes at roomtemperature in order to inactivate (that is, to inhibit) the Factor Xa.

Reversible Inactivation

Techniques for irreversibly and reversibly inactivating activated bloodfactors are disclosed in copending U.S. patent application Ser. No.08/268,003, filed Jun. 29, 1994, the pertinent parts of which are herebyincorporated by reference. Generally, reversible (that is, transient)inactivation may be accomplished by any of a variety of methods,including binding of an antibody/antibody fragment to the active region,binding of moiety which blocks sterically the proteolytic or otheractive domain, or incorporation of a chemical moiety which blocks theactive blood factor domain and gradually is released from the bloodfactor. In particularly preferred embodiments of this invention theblood factor is transiently inactivated by being acylated.

Reversible inactivation may be accomplished using benzamidines, whichare good reversible inhibitors of trypsin-like enzymes. The cationicamidino group of the inhibitor interacts with an enzyme carboxylatelocated at the bottom of the S1 subsite. A wide variety of substitutedbenzamidines have been investigated as inhibitors of thrombin andplasmin and are suitable for practice of this invention (see, e.g.,Andrews, J. M. et al, Jour. Med. Chem., 21:1202-07 (1978)). Extensivestudies have been reported on compounds containing two benzamidinemoieties, which are also desirable for the practice of this invention(see, e.g., Tidwell, R. R. et al., Thrombosis Research 19:339-49(1980)). 1,2-bis(5-amidino 2-benzofuranyl) ethane is also useful fortransient inhibition, and is known to inhibit Factor Xa with a Ki of 570Nm.

Also suitable for transient inactivation in the activation/inhibitionstep according to the invention are Kunitz inhibitors (a class of widelystudied protease inhibitors). Bovine pancreatic trypsin inhibitor(aprotinin) and tissue factor pathway inhibitor (also known as LACl)belong to this class. Dissociation constants (T) can range from 17 weeksto 11 seconds (Gebhard, W. et al., Proteinase Inhibitors, (1986)Elsevier). Aprotinin competitively inhibits factor VIIa with a Ki of 30uM (Chabbat, J. et al., Thrombosis Research, 71:205-15 (1993)).

Treatment to inhibit activated blood factors by acylation according tothe invention, proceeds by standard acylation reaction of thecorresponding blood factor, whether recombinantly produced or isolatedfrom plasma, according to procedures analogous to those set forth, forexample, or referenced in, Cassels, R. et al., Biochem. Jour.,247:395-400 (1987), or U.S. Pat. No. 4,337,244.

In certain embodiments in the activation/inhibition step according tothe invention, the partially purified preparation containing the bloodfactor is treated with a three to thirty-fold molar excess of anacylating agent in a neutral pH buffer at room temperature. Catalyticactivity is followed over a time course of approximately one to sixty,and preferably for ten to thirty minutes to assure the desired level ofinactivation of protein. The reagent is preferably prepared as a 0.1Msolution in DMSO or water and added to the protein at pH 7.5. Blockedprotein is subjected to chromatography (preferably on a gel-filtrationor ion-exchange column) at pH 5.0 to remove excess reagent. Protein maybe stored at pH 5.0 at -70° C. to -80° C. prior to further use.

Suitable active site acyl groups for use in this invention includebenzoyl, p or o methyl (toluoyl), p or o methoxy (p is a more preferredanisoyl), p or o fluoro benzoyl, Dimethyl acryloyl (3,3 or 3,4),Difluoro compounds, CH₃ CO benzene (acetyl gp), CH₃ CO NH benzene(acetanilide), p or o ethoxy (or other alkyl groups), and guanidinobenzoyl.

Suitable esters for use in this invention include the 4-toluoyl ester,the 3,3-dimethyl acrylyl ester, cyclohexylidineacetyl ester, thecyclohex-1-enecarbonyl ester, the 1-methylcyclohexylidineacetyl ester,the 4-aminobenzoyl ester, the p-anisic acid p-amidinophenyl ester, theo-anisic acid p-amidinoophenyl ester, the 3,4 dimethyl benzoic acidp-amidinophenyl ester, the benzoic acid p-amidinophenyl ester, the 3,3dimethylacrylic acid p-amidinophenyl ester, and the PDAEB(4-N-(2-N'-(3-(2-pyridyldithio)-propenyl)aminoethyl) amino benzoylester. In general, the acylating agent will be the activated form of anon-toxic acid which provides a saturated, unsaturated or aromatic 5- or6-carbon ring to which a carboxyl is substituted. The ring may containfurther substitutions, such as amino, alkoxy, alkyl, addition ringsystems, or any other non-interfering non-toxic substituent. For FactorX and other blood factors having a catalytically active serine domain,any compound capable of acylating the serine hydroxyl group or otherwiseblocking the serine catalytic domain in a reversible manner is suitablefor synthesis of the acylated blood factor. As described in U.S. Pat.No. 4,337,244, in general, either direct or inverse acylating agents canbe used. For direct acylating agents, the acylating moiety is itselfattracted to the catalytic site of the Factor Xa or other blood factor;in the inverse acylating approach, the leaving group is thus attracted.The acylated form of the blood factor is then purified from the reactionmixture using standard purification techniques, including dialysis,chromatography, selective extraction, and the like.

Potent acylating agents such as 3-alkoxy 4-chloroisocoumarins have beenreported for a variety of serine proteases (Harper, J. W. et al., Jour.Am. Chem. Soc., 106:7618-19 (1984); Harper, J. W. et al., Biochemistry,24:7200-13 (1985)), and are suitable for use in theactivating/inhibiting step according to the invention. The stability ofthe acyl enzymes are dependent on the alkoxy groups, small groups givingtransiently stable (T <2 h) acyl enzymes.

The compounds produced according to the processes of this inventionwhich serve as acylated blood factor diagnostics and/or pharmaceuticalsmust have an appropriate deacylation rate which assures an appropriateclearance time in vivo. The acylated proteins reactivate in a time,temperature and pH dependent manner. Typically, deacylation is faster at37° C. than at room temperature, and is faster at pH 8.0 than at pH 7.5.The deacylation rate can be measured as having a half-life of at least 5minutes in vitro in buffer using prothrombinase and/or clotting assays.Deacylation can be measured directly as described in R. A. G. Smith etal., Progress in Fibrinolysis, Vol. VII, pp. 227-31 (1985, ChurchillLivingstone). Prothrombinase and clotting assays are described in D. L.Wolf et al., Jour. Biol. Chem., 266:13726 (1991).

In certain preferred embodiments, deacylation of acyl Factor Xa iscarried out by incubation in a solution of appropriate pH and assayingaliquots in an amidolytic or clotting assay. The relative activity iscalculated as a percentage of equivalent amount of active Factor Xacarried through the same incubations. The preferred assay for acylFactor VIIa involves multiple steps. The acyl enzyme is incubated in theappropriate buffer at a protein concentration of 160 nM. At each timepoint, an aliquot is diluted to 0.16 nM and incubated with lipidatedtissue factor (0.25 nM) for 1 min at room temperature. The factorVIIa/Tissue Factor mixture is then used for activation of Factor X andresulting Factor Xa assayed in an amidolytic assay.

Purification Following the Activation and Inhibition Step

Following the step of treating the partially purified preparation toactivate and inhibit the blood factor, any of a variety of subsequentpurification techniques and combinations of techniques, known in the artand such as those discussed above, can be used to bring the inhibitedactivated blood factor to a final acceptable degree of purity.

In a particularly preferred embodiment, the product resulting from theactivation and inhibition step described above, namely inhibited FactorXa, is ultrafiltered at ambient temperature using a Millipore Viresolve™unit or equivalent process to further remove potential contaminants(e.g., viruses, IgG, RVV-X, Factor X, etc.). For a standard one squarefoot Millipore Viresolve™ membrane unit, for example, typically thecross flow rate is maintained at between 1.0-1.7 liter/minutes, whilethe permeate rate is controlled at between 5-60 ml/min. A one squarefoot Viresolve™ unit has enough membrane capacity to filter at least onegram of Inhibited Factor Xa at a concentration of approximately 0.7±0.3mg/ml, however other parameters are suitable for the practice of thisinvention. In order to achieve enhanced product recovery, afterfiltration is completed, the system should be rinsed more than two, andpreferably at least five times with at least 75 ml each time using theappropriate buffer used in the reaction, pH 6.0-7.5 (although it must berecognized that other buffers, volumes and timing may be desired for aparticular application).

The resulting permeate may then be directly loaded at ambient or otherconvenient temperature onto an anion exchange or other chromatographycolumn. In this particularly preferred embodiment, a DEAE Fractogelresin is utilized with a loading capacity of at least 8 mg product perml of resin. When working with permanently inactivated compounds, thecolumn is preequilibrated with phosphate buffer at pH 6.5 (or pH 5.0-5.5for reversibly inactivated compounds) containing less than 0.2M sodiumchloride. The sample mass appropriate to the column loading capacity isapplied and once application is complete, the column is washed with atleast 5 column volumes of phosphate buffer at pH 6.5 (or pH 5.0-5.5)containing no sodium chloride. A step change is made to wash the columnwith at least 5 column volumes of phosphate buffer at pH 6.5 (orpH5.0-5.5) containing 0.2M sodium chloride. The product is then elutedwith a step change to phosphate buffer at pH 6.5 (or pH 5.0-5.5)containing 0.3M sodium chloride.

If desired, the elution pool from the DEAE Fractogel column (or otherpurification step) can be incubated with the inhibitory agent a secondtime in order to reduce the level of residual Factor Xa (or other bloodfactor) which may co-purify with the Inhibited Factor Xa (or other bloodfactor).

The elution pool from the DEAE Fractogel column (or second inactivationstep) may then be concentrated and diafiltered if desired into the finalstorage buffer of choice using for example a Filtron ultrafiltrationsystem with 8 kDa MWCO Omega type membranes.

Therapeutic and Other Uses of the Blood Factors

When used in vivo for therapy, the blood factors of the subjectinvention are administered to the patient in therapeutically effectiveamounts (i.e. amounts that have desired therapeutic effect). They willnormally be administered parenterally. The dose and dosage regimen willdepend upon the degree of the coagulation disorder, the characteristicsof the particular activated or inhibited blood factor used, e.g., itstherapeutic index, the patient, and the patient's history.Advantageously the blood factor is administered in an acute caresetting, or continuously over a period of 1-2 weeks, or over a number ofyears intravenously to treat disorders in vasculature function.Optionally, the administration is made during the course of adjuncttherapy such as angiography, angioplasty, thrombolysis, stent placement,heart/valve/artery/venous surgery or transplant, combined cycles of pro-or anti-coagulant therapies including platelet aggregation inhibitors,or as part of therapeutic administration of other cardiovascularmodulatory agent.

For parenteral administration the blood factors will be formulated in aunit dosage injectable form (solution, suspension, emulsion) inassociation with a pharmaceutically acceptable parenteral vehicle. Suchvehicles are inherently nontoxic, and non-therapeutic. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and 5%human serum albumin. Nonaqueous vehicles such as fixed oils and ethyloleate can also be used. Liposomes may be used as carriers. The vehiclemay contain minor amounts of additives such as substances that enhanceisotonicity and chemical stability, e.g., buffers and preservatives. Theblood factors will typically be formulated in such vehicles atconcentrations of about 0.1 mg/ml to 100 mg/ml.

The blood factor compositions used in therapy are formulated and dosagesestablished in a fashion consistent with good medical practice takinginto account the disorder to be treated, the condition of the individualpatient, the site of delivery of the composition, the method ofadministration and other factors known to practitioners. The bloodfactor compositions are prepared for administration according to thedescription of preparation of blood factors, infra.

EXAMPLES

The Examples that follow illustrate the invention by specific referenceto production of Inhibited Factor X. The Examples are intended to beillustrative only, and do not limit the scope of the invention.

EXAMPLE 1 Preparation of Immunoaffinity Resin

In one example of the practice of this invention for the preparation ofan anti-Factor X immunoaffinity column, anti-Factor X monoclonalantibody, from crude mouse ascites, was purified via protein A,S-Sepharose Fast Flow and DEAE Fast Flow chromatography in sequentialsteps and was then buffer exchanged into 0.1M sodium carbonate buffer,pH 8.5 in preparation for linkage to Tresyl-activated Agarose(Schleicher and Schuell). 5 liters of antibody solution at 2 mg/ml waslinked for no less than 12 hours at 4° C. to an equal volume of preparedTresyl-activated resin. The mixture was continuously agitated in orderto maintain optimum contact between the resin and the antibody solution.After coupling, the supernatant was recovered from the resin slurry andassayed by an absorbance measurement for protein concentration. Theresin was then incubated for at least 12 hours at 4° C. with 0.1M Trisbuffer, pH 8.5, in order to block the remaining unreacted Tresyl siteson the activated resin. After blocking, the supernatant was againcollected and assayed by an absorbance measurement. The resin was thenwashed with 10 resin volumes of 20 mM Potassium phosphate buffer at pH7.0 containing 1.0M sodium chloride. The high salt wash was alsocollected and assayed for absorbance. The antibody binding efficiency ofthe process was determined to be greater than 98% when calculated takingthe difference between the known amount of antibody in the initialmixture and the mass of antibody recovered in the sum of the supernatantsamples and then dividing by the initial starting mass of antibody.Antigen dynamic binding capacity was determined by packing a onemilliliter column of the coupled resin and applying Factor X to thecolumn after it had been equilibrated with phosphate buffered saline.The Factor X was then eluted with 0.1M CAPS buffer, pH 10.5 andneutralized with 2M HEPES to pH 7.5 for absorbance measurement. Thedynamic Factor X binding capacity was determined to be at least 0.1±0.2mg Factor X per milliliter of resin.

EXAMPLE 2 Partial Purification of Factor X from a Natural Plasma Source

In one example of practice of this invention for purification of FactorX from a natural plasma source using an anti-Factor X immunoaffinitycolumn, a 50 ml sample from the wash fraction of a Factor IX affinitypurification step containing approximately 2.2 mg total protein per ml(based on a dye-binding total protein assay) of which 50% of the totalprotein was Factor X was directly applied to a 100 ml radial flow columncontaining anti-Factor X linked to Tresyl-activated Agarosepre-equilibrated with phosphate buffered saline. The load sample wasapplied at a flow rate of 20 ml per minute to provide a residence timeof at least 5 minutes. After the flow through peak returned to baselineby washing with phosphate buffered saline, the column was then washedwith phosphate buffered saline containing 0.5M sodium chloride for atleast 3 column volumes. The Factor X was then eluted with 0.1M CAPSbuffer containing 0.025M sodium chloride at pH 10.5 in approximately 2.5column volumes. The elution pool was assayed by absorbance measurement,total protein assay, and SDS-PAGE. The analysis showed that the elutionpool is predominantly a single band, with no detectable majorcontaminant bands and that less then 15% of the Factor X loaded flowedthrough the column when the column was loaded to at least 67% ofcapacity.

EXAMPLE 3 Activation and Inhibition of Factor X and Anion ExchangeChromatography of Reaction Mixture

In one example of practice of this invention for the conversion offactor X to the inhibited form of Factor Xa and the subsequentpurification, 11 mg of immunoaffinity purified Factor X were reacted atambient temperature for one hour with EGR-ck (Peptisyntha, Belgium;EGR-ck obtained from Calbiochem, San Diego, Calif.; from Bachem,Torrance, Calif.; or from Bachem AG, Switzerland have performedsimilarly) and RVV-X (Haemtech, Burlington, Vt.; RVV-X obtained fromERL, Indianapolis, Ind. has performed similarly) at a molar ratio of20:1 EGR-ck:Factor X and at a mass ratio of 1:250 RVV-X:Factor X,respectively, in the presence of 5 mM calcium chloride. The RVV-Xreaction was stopped by the addition of concentrated EDTA to 10 mM. Thereaction mixture was analyzed by Size Exclusion HPLC and a chromogenicassay specific for Factor Xa. The results showed that the reactionconverting Factor X to Factor Xa had proceeded to greater than 80%conversion, and that the residual Factor Xa was less than 400 nanogramper milliliter of solution.

The reaction mixture was then directly applied to a 1.2 ml DEAE column(Fractogel 650 M, E. Merck, Darmstadt, FRG; other anion exchange resins,e.g., A Fast Flow and DEAE Fast Flow, obtained from Pharmacia, Uppsala,Sweden; Poros Q, Poros PEI, Poros IIQ, obtained from PerSeptiveBiosystems, Boston, Mass.; etc., have performed similarly)pre-equilibrated with 20 mM sodium phosphate buffer, pH 6.5, containing0.2M sodium chloride. The flow through was washed to baseline with 20 mMsodium phosphate buffer, pH 6.5, and then the column was washed with atleast 5 column volumes each of 20 mM sodium phosphate buffer, pH 6.5,containing 0.15M, 0.2M and 0.25M sodium chloride. The Inhibited FactorXa was eluted with a step to 0.3M sodium chloride in 20 mM sodiumphosphate buffer, pH 6.5, in a total of 8 column volumes. The column wasthen washed with a high salt solution, 1.0M sodium chloride and thenstripped with 0.5N sodium hydroxide. Total protein assays and SDS-PAGEwere performed on all eluted fractions along with Size Exclusion HPLC,Reversed-phase HPLC and contaminant ELISA's on the elution pool. TheHPLC results indicated that the elution pool was greater than 89% pureby both HPLC methods. The total protein recovery of the step resulted ina 97% mass balance, indicating good recovery of all protein loaded ontothe column. The contaminant assays indicated that the DEAE step was ableto clear contaminants such as anti-Factor X IgG and RVV-X at levels atleast 500-fold. Additionally, the SDS-PAGE gels indicated that manycontaminating bands had been removed from the load sample during theflow through and wash steps prior to elution. This result indicates thatthe DEAE binding capacity for Inhibited Factor Xa was at least 6milligrams of Inhibited Factor Xa per ml of resin.

EXAMPLE 4 Ultrafiltration of Reaction Mixture using Millipore Viresolve™

In one example of practice of this invention for the ultrafiltration ofthe reaction mixture, a 70 kDa NMWCO small area module (Millipore,Viresolve™ 70, containing 0.01 ft² membrane area) was used. Factor X(14.5 ml at 0.5 milligram per ml, or 29 mg), was reacted with RVV-X andEGR-ck as described in Example 3 above. The reaction mixture was thenrecirculated over the small area module at a cross flow rate of 12 mlper minute with a peristaltic pump for 30 minutes to equilibrate thesystem. Several permeate flow rates, ranging from 0.15 to 1.0 ml perminute, were collected and tested by absorbance measurement at 280 nm.All permeate flow rates exhibited greater than 90% passage of proteinwhen a representative permeate sample was collected. Thus, for scale-uppurposes, a volume reduction experiment was conducted to examine totalprotein recovery upon passage of a fixed quantity of protein. A totalprotein recovery of greater than 80% was achieved with no wash stepsincorporated. Additional wash steps maya be included if desired toincrease recovery. This experiment showed that per square foot ofmembrane area, at least one gram of total protein (at a concentration of0.5 mg per ml) from the reaction mixture was processed with greater than80% recovery in a time between one-half hour and one hour.

EXAMPLE 5 Large-scale Production of Highly Purified EGR-Factor Xa:Preparation of Immunoaffinity Resin

Examples 5 through 10 illustrate a process according to this inventionfor the large-scale production of highly purified EGR-Factor Xa.Although each of these examples stand independently, they may also beunderstood as processes which occurred sequentially, with the productfrom each of Examples 5 through 9 being used for the steps described inthe following example.

To prepare an immunoaffinity resin specific for Factor X, ten grams of ahighly purified ascites-derived murine monoclonal anti-Factor X antibodywas coupled to 2.2 liters of Actigel™ ALD low substitution monoaldehydeactivated resin (Sterogene, Arcadia, Calif.). The coupling reaction wascarried out in an 180 mm Moduline™ column (Amicon, Beverly, Mass.), in0.1M sodium phosphate buffer, 0.1M sodium cyanoborohydride, pH 7.0, for20±4 hours at 2°-8° C. The mixture was maintained as a homogeneousslurry through the use of continuous agitation with an overhead mixer.After coupling, the resin was allowed to settle and the column effluentcollected and assayed for the presence of antibody. Less than 5% of theoriginal antibody was detected in the supernatant by an absorbancemeasurement at 280 nm. The resin was then washed with 20 liters of 0.1Msodium phosphate buffer, 0.5M sodium sequentially, with the product fromeach of Examples 5 through 9 being used for the steps described in thefollowing example.

To prepare an immunoaffinity resin specific for Factor X, ten grams of ahighly purified ascites-derived murine monoclonal anti-Factor X antibodywas coupled to 2.2 liters of Actigel™ ALD low substitution monoaldehydeactivated resin (Sterogone, Arcadia, Calif.). The coupling reaction wascarried out in an 180 mm Moduline™ column (Amicon. Beverly, Mass.), in0.1M sodium phosphate buffer. 0.1M sodium cyanoborohydride, pH 7.0, for20±4 hours at 2°-8° C. The mixture was maintained as a homogeneousslurry through the use of continuous agitation with an overhead mixer.After coupling, the resin was allowed to settle and the column effluentcollected and assayed for the presence of antibody. Less than 5% of theoriginal antibody was detected in the supernatant by an absorbancemeasurement at 280 mm. The resin was then washed with 20 liters of 0.1Msodium phosphate buffer, 0.5M sodium chloride, pH 7.0 to removenon-specifically bound protein. The remaining unlinked monoaldehydelinkage sites were blocked by recirculating 0.1M Ethanolamine, 0.1Msodium cyanoborohydride, pH 7.0, through the resin for approximately 6±2hours at 2°-8° C. The resin was then extensively washed and equilibrated(>40 liters) with 20 mM Tris buffer, 150 mM sodium chloride, pH 7.5, toremove all traces of sodium cyanoborohydride. After the final wash andequilibration, the column effluent showed no detectable levels of sodiumcyanoborohydride. The binding capacity of the resin for Factor X wasdetermined to be at least 100 μg/ml.

EXAMPLE 6 Large-scale Production of Highly Purified EGR-Factor Xa:Immunoaffinity Chromatography Purification

A Factor X-containing, solvent-detergent treated, partially-purifiedplasma fraction was obtained from a licensed plasma product manufacturer(Alpha Therapeutic Corporation, City of Industry, Calif.). This materialwas supplied pre-concentrated and frozen in a sodium citrate/sodiumchloride buffer system, pH 6.8. The total protein concentration wasmeasured at between 1.4 and 1.5 mg/ml using a Bradford dye-binding totalprotein assay (BioRad, Hercules. Calif.). The Factor X concentration wasmeasured at approximately 1.0±0.1 mg/ml based on the results of areversed-phase HPLC (RP-HPLC) assay and a Factor X deficient coagulationassay (1 Unit=10 μg Factor X), 1.3 liters of the Factor X-containingplasma fraction was thawed for 16-24 hours at 2°-8° C. and then filteredthrough a 0.2 μm filter (Millipak™ 20, Millipore, Bedford, Mass.) toremove any particulate matter. Based on the resin binding capacity, fourcycles of the immunoaffinity column were required to process the 1.3liters of Factor X-containing plasma fraction. Thus, the filtered FactorX-containing plasma fraction was divided into four roughly equal volumes(330 ml±20 ml) for application to the anti-Factor X immunoaffinitycolumn (prepared as detailed above). For each cycle, the immunoaffinitycolumn (at 2°-8° C.) was equilibrated with 3-5 column volumes of 20 mMTris buffer, 150 mM sodium chloride, pH 7.5. After equilibration, thefiltered Factor X-containing plasma fraction was then loaded at aresidence time of less than 5 minutes per column volume and the flowthrough peak washed to baseline with at least 7 additional columnvolumes of 20 mM Tris buffer, 150 mM sodium chloride, pH 7.5. The FactorX was eluted with a pH step using 0.1M CAPS buffer, 25 mM sodiumchloride pH 10.5. Each immunoaffinity elution pool (in approximately 2-4column volumes) was immediately titrated to pH 7.5±0.2 with 2.0M HEPESadded to 110±10 mM. Absorbance measurements and RP-HPLC confirmed that atotal of 1.03±0.03 grams of highly purified Factor X was recovered fromthe four cycles (236.5 mg, 249.4 mg, 291 mg and 250.5 mg for cycles onethrough four respectively). Thus, the cumulative yield of Factor X inthe elution pools for the immunoaffinity step was 78.0±5.0% and thetotal Factor X recovery, including Factor X which flowed through thecolumn and was not captured, was 88.0±5.0%. The four elution pools werethen stored at 2°-8° C. for one or two days prior to further processing.

EXAMPLE 7 Large-scale Production of Highly Purified EGR-Factor Xa:Filtration and Concentration of Immunoaffinity Elution Pool

Because the pools were stored at 2°-8° C. and to add another viralreduction step, the pH-neutralized immunoaffinity elution pools made asdescribed in Example 6 were filtered through a 0.04 μm filter(Sealkleen™ 0.5 ft², Pall Corporation, East Hills, N.Y.) at 2°-8° C. Thefiltered immunoaffinity elution pools were then concentrated, at 2°-8°C., to 1.0±0.1 mg/ml using a Minisette™ Ultrafiltration system (Filtron,Northborough, Mass.) holding four 0.75 ft² 8 kDa Omega-typeultrafiltration cassettes. The transmembrane pressure was maintained at15±1 psig, while the cross flow rate and filtrate rates were 2.56±0.2liters/minutes and 0.28±0.02 liters/minute throughout the course of theconcentration step. The final concentration of Factor X was verified byan absorbance measurement at 280 nm. No protein was detected in any ofthe filtrate samples tested, and the recovery of Factor X for these twosteps was thus greater than 99.0%.

EXAMPLE 8 Large-scale Production of Highly Purified EGR-Factor Xa:Activation/Inactivation of Factor X

The concentrated Factor X made as described in Example 7 wassimultaneously activated with Russell's Viper Venom Factor X activatingenzyme (RVV-X, Haematologic Technologies, Inc., Essex Junction, Vt.) bythe addition of RVV-X to a mass ratio of 250:1 (Factor X:RVV-X), andinactivated with Glu-Gly-Arg-chloromethyl ketone (EGR-ck, Peptisyntha,Brussels, Belgium) added to a molar ratio of 20:1 (EGR-ck:Factor X). Theactivation reaction was initiated by the addition of 1.0M calciumchloride to a final concentration of 5 mM and performed at ambient roomtemperature 21°±3° C. The activation reaction was stopped after one hourby the addition of 0.5M EDTA, pH 8.0 to a final concentration of 10 mM.The percent mass conversion of Factor X was greater than 95.0%, asdetermined by Size-Exclusion-HPLC (SE-HPLC). The final concentrations ofFactor X and EGR-Xa after the reaction by RP-HPLC were 0.07 mg/ml and0.9 mg/ml. respectively. The total mass of EGR-Xa recovered after thereaction step was approximately 882.0±45.0 mg. The post-reaction mixturewas held at 2°-8° C. overnight for further processing the next day.

EXAMPLE 9 Large-scale Production of Highly Purified EGR-Factor Xa: ViralReduction Step

In order to clear impurities and contaminants (e.g., virus, RVV-X, andIgG) the reaction mixture made as described in Example 8 wasultrafiltered through a 1 ft² 70 kDa nominal molecular weight cut-offultrafiltration module (Viresolve™, Millipore, Bedford, Mass.). Prior toultrafiltration, the post-reaction mixture was filtered through a 0.45μm microfilter (Coming, Corning, N.Y.). The filtered post-reactionmixture was then ultrafiltered at a starting cross flow rate of 0.76±0.1liters/minutes and eventually increased to a final cross flow rate ofapproximately 1.2±0.1 liters/minutes. The permeate flow rate initiallywas controlled at 16.0±1.0 ml/minute and then lowered to 8.0±1.0ml/minute in an attempt to increase recovery. The permeate wascontinuously tested for protein passage using absorbance measurementsand showed a relatively constant 50% passage over the course of the twohour filtration. After the total retained volume reached approximatelyone hold-up volume or 50±20 ml, the retentate was washed with 50±20 mlusing 0.1M CAPS, 25 mM sodium chloride, 110 mM HEPES, pH 7.5. thewashing was repeated six more times for a total of seven washes using atotal of 425 mls of wash buffer. The EGR-Xa recovery of this step wasgreater than 90% and the total protein mass balance was 100.0±5.0%.

EXAMPLE 10 Large-scale Production of Highly Purified EGR-Factor Xa:Anion-exchange Chromatography

In a final polishing step to minimize contaminants and impurities, theultrafiltered reaction mixture of Example 9 was then directly loadedonto a 5×20 cm XK chromatography column (Pharmacia, Piscataway, N.J.)containing 125±10 ml DEAE Fractogel™ 650 M anion-exchange resin (E.M.Science, Gibbstown, N.J.). The column was pre-equilibrated with 5-10column volumes of 20 mM Potassium phosphate buffer, 0.2M sodiumchloride, 10 mM EDTA, pH 6.5. After completion of the sample load, thecolumn was first washed with 20 mM Potassium phosphate buffer, 10 mMEDTA, pH 6.5, until the flow-through peak had returned to within 10% ofbaseline. The column was then further washed with 20 mM Potassiumphosphate buffer, 0.2M sodium chloride, 10 mM EDTA, pH 6.5, for 5-10column volumes. The EGR-Xa was then eluted using 20 mM Potassiumphosphate buffer, 0.3M sodium chloride, 10 mM EDTA, pH 6.5. The elutionpeak was collected in 8±1 column volumes. The elution pool was assayedfor total protein concentration (Bradford and absorbance measurements),purity (RP-HPLC), residual Factor X and Xa (RP-HPLC and chromogenic,respectively), and residual RVV-X and Anti-Factor X IgG (ELISA).Approximately 750±50 mg of total protein was recovered, containinggreater than 95% EGR-Xa (a and β forms in a 30 to 1 ratio), less than 4%Factor X and less than 150 parts per million residual Factor Xa, lessthan 10 parts per million RVV-X and undetectable levels of Anti-Factor XIgG.

Table 1 summarizes the individual Factor X/EGR-Xa step yields andoverall process yield for the production of EGR-Xa from a partiallypurified Factor X-containing plasma fraction, as described in Examples10-15. The resulting EGR-Xa product showed greater than 100% clotinhibiting activity in an in vitro clotting assay (aPTT) when comparedto a commercially available EGR-Xa standard (Haematologic Technologies,Inc., Essex Junction, Vt.).

                  TABLE 1                                                         ______________________________________                                        Factor X Process Yield Summary                                                Process steps        Yield                                                    ______________________________________                                        Immunoaffinity capture efficiency                                                                   80.0 ± 10.0%                                         (overall Factor X mass balance                                                approximately 75-85%)                                                         Immunoaffinity step yield                                                                          78.0 ± 5.0%                                           Filtration/Concentration step yield                                                                99.0 ± 5.0%                                           Reaction step yield  89.5 ± 5.0%                                           Viral filtration step yield                                                                        90.7 ± 5.0%                                           Anion-exchange step yield                                                                          93.8 ± 5.0%                                           Overall process yield                                                                               58.0 ± 10.0%                                         ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 306 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 59..64                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 79..95                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 160                                                             (D) OTHER INFORMATION: /note= "Disulfide linkage to                           residue 132 of SEQ ID NO:2"                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 208..222                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 233..261                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerValAlaGlnAlaThrSerSerSerGlyGluAlaProAspSerIle                              151015                                                                        ThrTrpLysProTyrAspAlaAlaAspLeuAspProThrGluAsnPro                              202530                                                                        PheAspLeuLeuAspPheAsnGlnThrGlnProGluArgGlyAspAsn                              354045                                                                        AsnLeuThrArgIleValGlyGlyGlnGluCysLysAspGlyGluCys                              505560                                                                        ProTrpGlnAlaLeuLeuIleAsnGluGluAsnGluGlyPheCysGly                              65707580                                                                      GlyThrIleLeuSerGluPheTyrIleLeuThrAlaAlaHisCysLeu                              859095                                                                        TyrGlnAlaLysArgPheLysValArgValGlyAspArgAsnThrGlu                              100105110                                                                     GlnGluGluGlyGlyGluAlaValHisGluValGluValValIleLys                              115120125                                                                     HisAsnArgPheThrLysGluThrTyrAspPheAspIleAlaValLeu                              130135140                                                                     ArgLeuLysThrProIleThrPheArgMetAsnValAlaProAlaCys                              145150155160                                                                  LeuProGluArgAspTrpAlaGluSerThrLeuMetThrGlnLysThr                              165170175                                                                     GlyIleValSerGlyPheGlyArgThrHisGluLysGlyArgGlnSer                              180185190                                                                     ThrArgLeuLysMetLeuGluValProTyrValAspArgAsnSerCys                              195200205                                                                     LysLeuSerSerSerPheIleIleThrGlnAsnMetPheCysAlaGly                              210215220                                                                     TyrAspThrLysGlnGluAspAlaCysGlnGlyAspSerGlyGlyPro                              225230235240                                                                  HisValThrArgPheLysAspThrTyrPheValThrGlyIleValSer                              245250255                                                                     TrpGlyGluGlyCysAlaArgLysGlyLysTyrGlyIleTyrThrLys                              260265270                                                                     ValThrAlaPheLeuLysTrpIleAspArgSerMetLysThrArgGly                              275280285                                                                     LeuProLysAlaLysSerHisAlaProGluValIleThrSerSerPro                              290295300                                                                     LeuLys                                                                        305                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 139 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 17..22                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 50..61                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 55..70                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 72..81                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 89..100                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 96..109                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 111..124                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 132                                                             (D) OTHER INFORMATION: /note= "Disulfide linkage with                         residue 160 of SEQ ID NO:1, residue 108 of SEQ ID NO:3 or                     residue 108 of SEQ ID:4"                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaAsnSerPheLeuThrThrMetLysLysGlyHisLeuThrArgThr                              151015                                                                        CysMetThrThrThrCysSerTyrThrThrAlaArgThrValPheThr                              202530                                                                        AspSerAspLysThrAsnThrPheTrpAsnLysTyrLysAspGlyAsp                              354045                                                                        GlnCysGluThrSerProCysGlnAsnGlnGlyLysCysLysAsxGly                              505560                                                                        LeuGlyGluTyrThrCysThrCysLeuGluGlyPheGluGlyLysAsn                              65707580                                                                      CysGluLeuPheThrArgLysLeuCysSerLeuAspAsnGlyAspCys                              859095                                                                        AspGlnPheCysHisGluGluGlnAsnSerValValCysSerCysAla                              100105110                                                                     ArgGlyTyrThrLeuAlaAspAsnGlyLysAlaCysIleProThrGly                              115120125                                                                     ProTyrProCysGlyLysGlnThrLeuGluArg                                             130135                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 254 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 7..12                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 27..43                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 108                                                             (D) OTHER INFORMATION: /note= "Disulfide linkage with                         residue 132 of SEQ ID NO:2"                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 156..170                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 181..209                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       IleValGlyGlyGlnGluCysLysAspGlyGluCysProTrpGlnAla                              151015                                                                        LeuLeuIleAsnGluGluAsnGluGlyPheCysGlyGlyThrIleLeu                              202530                                                                        SerGluPheTyrIleLeuThrAlaAlaHisCysLeuTyrGlnAlaLys                              354045                                                                        ArgPheLysValArgValGlyAspArgAsnThrGluGlnGluGluGly                              505560                                                                        GlyGluAlaValHisGluValGluValValIleLysHisAsnArgPhe                              65707580                                                                      ThrLysGluThrTyrAspPheAspIleAlaValLeuArgLeuLysThr                              859095                                                                        ProIleThrPheArgMetAsnValAlaProAlaCysLeuProGluArg                              100105110                                                                     AspTrpAlaGluSerThrLeuMetThrGlnLysThrGlyIleValSer                              115120125                                                                     GlyPheGlyArgThrHisGluLysGlyArgGlnSerThrArgLeuLys                              130135140                                                                     MetLeuGluValProTyrValAspArgAsnSerCysLysLeuSerSer                              145150155160                                                                  SerPheIleIleThrGlnAsnMetPheCysAlaGlyTyrAspThrLys                              165170175                                                                     GlnGluAspAlaCysGlnGlyAspSerGlyGlyProHisValThrArg                              180185190                                                                     PheLysAspThrTyrPheValThrGlyIleValSerTrpGlyGluGly                              195200205                                                                     CysAlaArgLysGlyLysTyrGlyIleTyrThrLysValThrAlaPhe                              210215220                                                                     LeuLysTrpIleAspArgSerMetLysThrArgGlyLeuProLysAla                              225230235240                                                                  LysSerHisAlaProGluValIleThrSerSerProLeuLys                                    245250                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 241 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 7..12                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 27..43                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 108                                                             (D) OTHER INFORMATION: /note= "Disulfide linkage with                         residue 132 of SEQ ID NO:2"                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 156..170                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 181..209                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       IleValGlyGlyGlnGluCysLysAspGlyGluCysProTrpGlnAla                              151015                                                                        LeuLeuIleAsnGluGluAsnGluGlyPheCysGlyGlyThrIleLeu                              202530                                                                        SerGluPheTyrIleLeuThrAlaAlaHisCysLeuTyrGlnAlaLys                              354045                                                                        ArgPheLysValArgValGlyAspArgAsnThrGluGlnGluGluGly                              505560                                                                        GlyGluAlaValHisGluValGluValValIleLysHisAsnArgPhe                              65707580                                                                      ThrLysGluThrTyrAspPheAspIleAlaValLeuArgLeuLysThr                              859095                                                                        ProIleThrPheArgMetAsnValAlaProAlaCysLeuProGluArg                              100105110                                                                     AspTrpAlaGluSerThrLeuMetThrGlnLysThrGlyIleValSer                              115120125                                                                     GlyPheGlyArgThrHisGluLysGlyArgGlnSerThrArgLeuLys                              130135140                                                                     MetLeuGluValProTyrValAspArgAsnSerCysLysLeuSerSer                              145150155160                                                                  SerPheIleIleThrGlnAsnMetPheCysAlaGlyTyrAspThrLys                              165170175                                                                     GlnGluAspAlaCysGlnGlyAspSerGlyGlyProHisValThrArg                              180185190                                                                     PheLysAspThrTyrPheValThrGlyIleValSerTrpGlyGluGly                              195200205                                                                     CysAlaArgLysGlyLysTyrGlyIleTyrThrLysValThrAlaPhe                              210215220                                                                     LeuLysTrpIleAspArgSerMetLysThrArgGlyLeuProLysAla                              225230235240                                                                  Lys                                                                           __________________________________________________________________________

I claim:
 1. A process for preparing an inhibited form of an activated blood factor, comprising the steps of providing a partially purified preparation containing blood factor Factor VII, treating the partially purified preparation to convert the blood factor to an activated blood factor and to convert the activated blood factor to an inhibited form in a single step, and then recovering the resulting inhibited activated blood factor.
 2. The process of claim 1 wherein said step of treating the partially purified preparation comprises reacting the preparation with a blood factor activating agent and reacting the preparation with an activated blood factor inhibiting agent.
 3. The process of claim 2 wherein said reacting the preparation with a blood factor activating agent and said reacting the preparation with an activated blood factor inhibiting agent are carried out concurrently.
 4. The process of claim 2 wherein said reacting the preparation with an activated blood factor inhibiting agent is carded out before said reacting the preparation with a blood factor activating agent is carried out.
 5. The process of claim 2 wherein said reacting the preparation with an activated blood factor inhibiting agent is carried out after said reacting the preparation with a blood factor activating agent is carried out.
 6. A process for producing a highly purified preparation of an inhibited form of an activated blood factor Factor VII, comprising the steps of providing a partially purified preparation containing the blood factor, treating the partially purified preparation to convert the blood factor to an activated blood factor and to convert the activated blood factor to an inhibited form in a single reaction vessel, and then recovering the resulting inhibited activated blood factor.
 7. The process of claim 6, wherein said conversion to an activated blood factor and said conversion to an inhibited form are carried out without intervening process steps.
 8. The process of claim 6, wherein said inhibited activated blood factor is recovered at the level of purity suitable for pharmaceutical administration.
 9. The process of claim 6, wherein the inhibited activated blood factor is recovered using immunoaffinity chromatography utilizing an antigen-specific monoclonal antibody coupled to an activated resin selected from the group consisting of: agarose, cross-linked agarose, dextran, cross-linked polysaccharide, polymethyl methacrylate, and synthetic polymeric-based resin.
 10. The process of claim 6, wherein the inhibited activated blood factor is recovered using immunoaffinity chromatography utilizing an antigen-specific monoclonal antibody coupled to an activated resin, and wherein the activated resin utilizes an activation chemistry selected from the group consisting of: tresyl, azlactone, aldehyde, hydrazide, N-hydroxyl succinimide and triazine.
 11. The process of claim 6, wherein the inhibited activated blood factor is recovered using an anion exchange column having an anion exchange group linked to a naturally derived polysaccharide or a synthetically derived polymeric matrix.
 12. The process of claim 6, wherein said partially purified blood factor is treated with an activating enzyme in solution.
 13. The process of claim 6, wherein said partially purified blood factor is treated with an immobilized activating enzyme. 